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O P Samarina

Publications and source records attributed to O P Samarina.

16 recordsLinked to original sources
PubMed1979

Cross-linked informofers.

The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethyl-suberimidate and dimethyl-3,3'-dithiobispropionimidate). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linked reagent had been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or poly-particles) were reconstituted upon mixing of cross-linked informofers with pre-mRNA and removal of 2 M NaCl.

Animals↗
PubMed1979

Hybridization properties of sequences adjacent to triphosphorylated 5'-ends of nuclear pre-mRNA from mouse Ehrlich carcinoma.

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method /1/. These fragments as well as fragments of total pre-mRNA of the same size were polyadenylated in vitro by ATP:RNA adenyltransferase and used as templates for the synthesis of [32P] cDNA by reverse transcriptase in the presence of an oligo(dT) primer. The use of cDNA transcribed from the triphosphorylated 5'-end fragments of pre-mRNA (5'-cDNA) and from the total pre-mRNA fragments allows one to calculate the complexity of the 5'-end fraction pre-mRNA and to detect these sequences in polysomal mRNA. Sequences adjacent to 5'-phosphorylated ends of pre-mRNA represent a specific class of sequences with a complexity of about 200 kb. It was also found that about 25% of total pre-mRNA and about a half of sequences adjacent to triphosphorylated 5'-ends are present in polysomal mRNA. A high homology between triphosphorylated 5'-end fragments of pre-mRNA and mRNA sequences may be explained in terms of splicing. Less than 30% of 5'-cDNA hybridized to moderately repetitive DNA while most of them are represented by unique DNA sequences. About 15% of 5'-cDNA contained oligo(dA) sequences originated from oligo(U) in pre-mRNA from which it was transcribed.

Animals↗
PubMed1976

The properties of oligonucleotide fragments containing the triphosphorylated 5'-termini of nuclear pre-mRNA from Ehrlich ascites carcinoma cells.

Triphosphorylated 5'-end fragments 50-150 nucleotides in length were isolated from nuclear pre-mRNA with the aid of a hydroxyapatite chromatography. They are enriched in U and G (28 and 26%, respectively). About 15% of the fragments isolated from poly(U)+RNA contain poly(U) tracks. Neither poly(A)- nor double-stranded sequences were found. Hybridization experiments in conditions of vast DNA excess demonstrated that the 5'-end fragments contain a low amount of highly repetitive sequences but enriched in sequences hybridizing at C0t 1/2 approximately 100.

Animals↗
PubMed1976

The existence of triphosphorylated 5'-ends in virus-specific RNA isolated from SV-40 transformed cells.

The question about the nature of promoters in the transcriptional units containing SV-40 sequences in transformed cells was analyzed. It was found that the pulse-labeled RNA hybridizing to SV-40 DNA contains small but significant amounts of triphosphorylated 5'-ends detected as pppGp in alkaline hydrolyzates of this RNA. In another series of experiments the fragments of RNA containing triphosphorylated 5'-ends about 100 nucleotides in length have been isolated by hydroxyapatite chromatography. Some of them form hybrids with SV-40 DNA. The conclusion is drawn that at least some of SV-40 promoters are used for transcription initiation in SV-40 transformed cells.

Alkaline Phosphatase↗
PubMed1976

Autodegradation of pre-mRNA containing nuclear ribo-nucleoprotein particles. The effect of autodegradation on the double-stranded RNA sequences and on the protein composition of particles.

The quantitative changes of double-stranded RNA components of nuclear ribonucleo-protein particles containing pre-mRNA was investigated in the course of incubation of particles at 37 degrees C. The incubation of purified nuclear particles revealed the fragmentation of long double-stranded RNA sequences into shorter stretches. The presence of nuclear sap in the incubation mixture resulted in degradation of the double-stranded RNAs into acid soluble products. Autodegradation and/or ribonuclease treatment of nuclear RNP particles is accompanied by quantitative changes in the minor protein constituents of informofer.

Animals↗
PubMed1976

On the nature of 5' termini in nuclear pre-mRNA of Ehrlich carcinoma cells.

5' terminal nucleosides of nuclear pre-mRNA of Ehrlich ascites carcinoma cells were analyzed by a combination of different chromatographic methods and phosphatase treatment. The heavy nuclear pre-mRNA contains mainly unblocked triphosphorylated nucleosides at the 5' end, although some capped 5' ends could also be found. In this respect, it differs from cytoplasmic poly(A)+ mRNA which contains blocked 5' termini and no triphosphorylated ends. The 5' terminal nucleotides in pre-mRNA are pppGp and pppAp (in a ratio of 3:2). The determination of pppNp content in poly (A)+, poly(U)+, and poly (A)-(U)- fragments of RNA has been used as an approach to establish the topography of pre-mRNA. We also established that the technique for isolation of triphosphorylated 5' terminal fragments of RNA based on hydroxyapatite chromatography (Bajszár, Samarina, and Georgiev, 1974) is still valid in the presence of blocked oligonucleotides. The latter do not interfere with fragments containing free triphosphate groups. Using this technique, we showed that a small but significant portion of triphosphorylated 5' end fragments of 100 nucleotides in length contain oligo(U) sequences reacting with poly(A)-Sepharose.

Base Sequence↗
PubMed1976

The poly(A)-containing ribonucleoproteins in the nucleus and the cytoplasm of ehrlich ascites carcinoma cells.

The sedimentation characteristics and protein composition of the polyadenylic acid-containing ribonucleoprotein (RNP) particles separated from the polysomes of Ehrlich ascites carcinoma cells by means of ribonuclease treatment were studied. The results were compared with the corresponding characteristics of the poly(A)-containing particles of the cell nucleus. The sedimentation coefficient of the poly(A)-containing RNP from poly-ribosomes was found to be 11 S, while that of the nuclear particles was 14 S. In Cs2SO4 equilibrium density gradient values of p=1.25 and 1.34 gcm-3 were obtained for the nuclear particles and the poly(A)-RNP separated from polyribosomes, respectively. The molecular weights of the proteins making up the polysomal poly(A)-particles were found to be over 40 000 daltons, i.e. 54 000, 48 000, 68 000 and 89 000 daltons for the four polypeptides present in considerable amounts. As far as electrophoretic mobility is concerned these proteins were indistinguishable from the proteins of the nuclear poly(A)-containing RNP. However, the ratio of the components in the two types of particle proved to be quite different.

Animals↗
PubMed1975

DNA-synthesis on giant nuclear RNA by AMV DNA polymerase.

The reverse transcription of pre-mRNA isolated from rat liver or mouse Ehrlich ascites carcinoma cells with the aid of hot phenol fractionation technique is described. Pre-mRNA isolated at 85 degrees C is a more active template than the 65 degree C fraction. The addition of oligo(dT) as a primer strongly stimulated the template activity of the 65 degree C fraction. The size of product corresponds to a sedimentation value of 7 S as measured in alkaline sucrose gradient and is essentially less than the size of template.

Animals↗
PubMed1975

Protein composition of nuclear 14 S ribonucleoprotein particles containing poly (A).

Nuclear 14 S RNP particles containing poly (A) from Ehrlich ascites carcinoma cells and rat liver were purified by re-sedimentation in sucrose gradients, by Cs2SO4 density gradient centrifugation and by affinity chromatography on a poly (dT)-Sepharose column. Proteins of these RNP particles were electrophoresed in urea and SDS-polyacrylamide gels. RNP particles of ascites carcinoma cells contain two main bands having molecular weights of 51 000 and 69 000 daltons, respectively, and two or three minor components.

Adenine Nucleotides↗
PubMed1975

Purification of nuclear ribonucleoprotein complexes containing poly(adenylic acid).

Nuclear 14 S ribonucleoprotein particles containing poly(adenylic acid) were isolated from Ehrlich ascites carcinoma cells and purified by a two step method. The nuclear extract obtained at an ionic strength of 0.1 was treated by pancreatic and T1 ribonucleases and sedimented in a 15-30% sucrose gradient. The 14 S peak containing 50-60% poly(adenylic acid) was chromatographed on a poly(dT)-Sepharose column. The particles thus obtained were of high purity: they had an adenosine content of 90% and a protein moiety characteristic of the 14 S nuclear particles i.e. polypeptides with molecular weight of 51 000, 61 000 and 69 000 daltons.

Animals↗
PubMedDate not supplied

[Nuclear ribonucleoproteins containing pro-mRNA. XIV. Structural study using ethidium and fluorescamine].

Nuclear 30S RNP particles were studied by means of fluorescence techniques. It's shown that fluorescamin interacts with NH2-groups of protein molecule. As a result, covalent fluorescent label is formed. Quantum yield (rho), fluorescence spectra, lifetime of excited state (tau) and polarization of fluorescamin complexes with 30S particles were studied. Excitation spectra have their maximum at 395 nm, and fluorescence spectrum at 480 nm. These figures correspond to spectra of fluorescamin complexes with NH2-groups of lysine. Mean quantum yield (rho = 0.27) and lifetime of excited state of fluorescence (tau = 7.8 nsec) were measured. It's shown that fluorescamin forms two types of fluorescent complexes in 30S particles. These complexes differ only by their rho(rho1 = 0.11, rho2 = 0.30) and rho(rho1 = 3.6 nsec, rho2 = 10.0 nsec) by 2.7 times. Migration radius between fluorescamin bound to protein and ethydium bromide adsorbed on double-stranded regions of pre-mRNA in RNP-particles was measured. It's equal to 32 A. Adsorbtion isotherms of ethydium bromide were measured by fluorescence in 0.1 and 0.4 M NaCl. Data obtained showed that 6% of pre-mRNA in 30S particles bound the dye as a strong complex, i. e. this part of pre-mRNA is double-stranded. RNase treatment of RNP had no effect on this value. But the increase of NaCl concentration up to 0.4 M caused the dissociation of protein subunits to some extent followed by appearance of up to 40% free NH2-groups interacting with fluorescamin. Measuring of energy migration from fluorescamin to ethydium bromide showed that double-stranded pre-mRNA regions strictly bound to protein sticked out from RNP particle at a distance of about 27 A. The increase of NaCl concentration up to 0.4 M leads to disruption of this strict bond of double-stranded regions with protein. As a result, these regions of pre-mRNA become labile and move away from the RNP particle at more than 30 A. According to theoretical calculations, there is about 1--2 pre-mRNA hairpins (18--9 base pairs respectively) per one 30S particle.

Ethidium↗
PubMedDate not supplied

[Nuclear RNP, containing pre-mRNA. XIV. Nature of particles, containing 5'- and 3'-end structures].

Some characteristic peculiarities of the 5'-end and 3'-end structures of pre-mRNA isolated from nuclear RNP particles have been investigated: presence of triphosphorylated nucleotides on the 5'-ends, as a characteristic of the primary product of transcription; presence of modified 5'-ends -- blocked and methylated structures -- caps; presence of poly(A) blocks attached to the 3'-end of pre-mRNA during post-transcriptional transformation of the latter. It was shown that pre-mRNA isolated from nuclear RNP particles contained triphosphorylated nucleotides as well as a "cap" structure at the 5'-ends. On the 3'-ends of pre-mRNA from nuclear RNP particles isolated in the presence of a RNAse inhibitor, the presence of poly(A) blocks have been shown. These poly(A) structures are separated very easily from pre-mRNA during mild RNAase digestion. This is the reason why they are not detected in 30S monoparticles, containing pre-mRNA fragments connected with one informofer. Almost all poly(A) complexes in this condition are combined with proteins and have the sedimentation coefficient of about 14S. It was concluded that formation of nuclear pre-mRNA containing RNP-particles take place just after the onset of RNA synthesis. All processing steps occur with pre-mRNA packed in nuclear RNP particles.

Animals↗
PubMedDate not supplied

[Structure of nuclear pre-mRNA. XI. Triphosphorylated and blocked 5'-ends in the pre-mRNA].

The nature of the 5'-termini in pre-mRNA isolated from Ehrlich carcinoma cells has been investigated. To discriminate between triphosphorylated 5'-ends and capped structures different methods were used including treatment by alkaline phosphatase and several chromatographic methods. It was shown that heavey pre-mRNA contains a significant number of non-blocked triphosphorylated nucleotides at the 5'-end termini. However, phosphatase resistent, blocked 5'-termini were also found. 5'-terminal nucleotides in triphosphorylated pre-mRNA are G in a 3 : 2 ratio. In contrast to nuclear pre-mRNA cytoplasmic poly(A)+mRNA does not contain triphosphorylated 5'-ends but does contain the "cap" structure only. To elucidate the pre-mRNA topography the localization of homopolymeric regions of pre-mRNA, poly(A) and oligo(U), in relation to 5'terminal structures has been investigated. The experiments showed that the distance between 3'-terminal poly(A) sequences and 5'-end triphosphates is longer than 1500--2000 nucleotides. At the same time the distance between the latter and oligo(U) in pre-mRNA is much shorter.

Alkaline Phosphatase↗
PubMedDate not supplied

[DNA synthesis on the heterogeneous nuclear RNA template catalysed by DNA polymerase of avian myeloblastosis virus].

Template activity of nuclear pre-mRNA has been investigated in DNA-polymerase reaction. Active synthesis of DNA was demonstrated on pre-mRNA as a template in the absence of primer. A part of synthetic activity may be attributed to the traces of DNA present in the pre-mRNA preparation. Addition of oligo(dT)10 to the template stimulated the synthesis of DNA product due to transcription of heteropolymeric regions near the poly(A). The rate of DNA synthesis was different depending on the fraction of template used: the RNA extracted by hot phenol at 85 degrees showed higher template activity without adding of primer than the 65 degrees C fraction. On the contrary 65 degrees C pre-mRNA which is known to contain greater quantity of molecules with poly(A) at the 3'-end is more strongly stimulated by addition of oligo(dT). The nuclear RNA corresponding to the precursors of rRNAs extracted at 40 degrees C were not transcribed by the reverse transcriptase. The size of the DNA-product (about 7-8S in alkaline sucrose gradient) did not depend on the size of the template neither on the presence of oligo(dT)10 primer. The inhibition of the second DNA strand synthesis with actinomycin D had also no influence on the size of DNA-product.

Avian Leukosis Virus↗
PubMedDate not supplied

[Nuclear ribonucleoproteins containing messenger RNA. 12. Fluorescence studies of the secondary structure of pre-mRNA in nuclear RNP-particles].

Secondary structure of pre-mRNA in nuclear ribonucleoprotein particles (30S-particles) was examined using fluorescent dyes: acridine orange, acriflavine and ethidium bromide. Comparison of ethidium bromide and acriflavine adsorption isotherms for RNP-particles and free RNA and a study of acridine orange dimerization on binding to RNP revealed that 70% of pre-mRNA in 30S-particles is accessible for the dye binding. Dye molecules were adsorbed on double-stranded sequences (11--12% of the total amount of RNA in 30S-particles) and on the single-stranded parts of RNA (58--59% of 30S-particles), the rest part of RNA was unaccessible for the dye binding. A method involving measurements of acriflavine fluorescence quantum yields was used for the determination of nucleotide composition of double-stranded parts of RNA in the 30S-particles. AU-nucleotide content thus obtained was approximately 50%, as was established also for free pre-mRNA. Na+ ions weaken the interaction between the protein and pre-mRNA in 30S particles and increase mobility of double-stranded parts of this nucleic acid.

Animals↗